The development of genetic vectors that can deliver therapeutic DNA sequences to the central nervous system (CNS) in situ promises to be a powerful means of intervention in neurologic diseases, many of which have not current form of therapy. Viral-based vectors currently provide the most efficient means of gene delivery to the CNS. The goal of gene therapy is stable, long term expression of the introduced transgene. Each viral based system has relative advantages, however none of the current generation of vectors has maintained sustained expression in a significant proportion of target cells. Although transgenes are targeted to neurons and glia, the fate of transgene expression after introduction ot the cell is invariably reduced. This proposal concerns the rational development of a vector system for the CNS that can efficiently traduce post-mitotic cells, with stable expression over long periods. This vector is a plasmid based amplicon that contains elements from both herpes simplex virus type 1 (HSV-1) and adeno- associated virus (AAV). This hybrid vector is designed to carry large or multiple transgenes, to target efficiently to the nucleus of neurons, and to integrate efficiently in a site specific manner into a non-essential locus in the human genome. The vector constructs will be engineered to maximize the efficiency and specificity of integration in dividing and post-mitotic cells. Analysis of vector-transduced cells by Southern blots of genomic DNA, PCR and nucleotide sequencing will map integration at the population and clonal cell levels. We will test the stability of integration and correlate the event with long term expression. Further, we propose to test this vector in a novel in vivo platform for human gene therapy in the CNS. The human cell line, NT2N forms stable xenografts, differentiating into apparently mature glutamatergic neurons in the caudate-putamen of immunocompromised rats. This system will be exploited to test the time course of expression, integration, toxicity and delivery of genes in vivo. Taken together, this proposal describes the development of CNS vectors that promote the long term expression of transgenes by their introduction into a stable genetic environment within the neuron. This is intended to shed light on the genetic requirements for stable expression in the postmitotic adult brain. Stable expression remains the limiting factor in the introduction of therapeutic genes into the CNS for the treatment of a myriad of neurological diseases.